Affinity chromatography is a separation technique which is based on the specific binding properties of biological molecules. Briefly, the methodology of affinity chromatography involves the attachment of a specific ligand to an insoluble support or matrix to form a conjugate which is then contacted with a "feed" containing the substance to be purified either in a column or batch configuration. Contact of the feed with the conjugate results in the substance that reacts specifically with the ligand becoming attached to the conjugate with all the remaining components of the feed passing through the column in the void volume. The adsorbed substance is then eluted from the column by imposing conditions which dissociate it from the conjugated ligand. Preferably, the column can then be recycled and the affinity adsorbent reused for additional purifications.
Generally speaking, an affinity chromatography system has several components; a support for attaching the ligand, which is generally made from a polysaccharide but may also be made from other materials such as polyacrylamide gel, silica, other polymers or glass; optionally a spacer or "arm" between the ligand or support used to contribute to the binding of macromolecules such as proteins to affinity columns; and the ligand which is specifically designed based on its capability to bind to the substance to be purified. The ligand may be an enzyme, co-enzyme, the substrate or inhibitor of an enzyme, an antibody, an antigen, and the like.
With respect to the support components of the affinity chromatography system, a number of materials suited for use with multiple ligands are commercially available. Most commonly, the ligand is covalently bound to the support and for this purpose so called "activated" supports which are pre-configured for covalent linkage with ligands are commercially available.
In order for the affinity chromatography system to be recycled it is desirable that the support and ligand be resistant to breakdown during the purification process. However, severe shortcomings are encountered as a result of the instability of the covalent chemical linkages between the support and the ligand. Polysaccharide supports such as agarose using covalently coupled ligands are susceptible to microbial attack and suffer from instability with respect to mechanical destruction and "ligand leakage" on prolonged washing. When the ligand is a polysaccharide (for example chondroitin sulfate) these same problems of microbial attack, mechanical destruction and "ligand leakage" become important considerations. When the affinity chromatography system uses an enzyme substrate as a ligand which is covalently bound to the support, the ligand is highly susceptible to breakdown by the enzyme during the purification process destroying the integrity of the support-ligand attachment which requires replacement of the affinity chromatography system. Repeated cycling of the covalently coupled system invariably results in a lowering of binding capacity and in extreme cases, binding capacity can be totally lost due to cleavage of the polysaccharide ligand by the enzyme or by microbial attack. Due to the high cost of the so called "activated" matrix supports which utilize covalent coupling, replacement of the systems becomes an expensive drawback to the affinity chromatography purification technique.
There is thus a need for an affinity chromatography system which avoids the shortcomings and expense associated with conventional covalently coupled systems and allows replacement of the ligand to be accomplished simply and without the need to replace the entire matrix support system.